Chemokine receptor antagonist and cyclosporin in combined therapy

ABSTRACT

The use of a chemokine receptor antagonist together with a cyclosporin to produce a pharmaceutical composition for treating or preventing rejection of transplanted organs, tissues or cells is herein disclosed. Said pharmaceutical compositions for the simultaneous, sepatate or sequential use of its active ingredients for the above specified therapy are also disclosed and claimed. In particular, the use of Met-RANTES together with cyclosporin A to produce a pharmaceutical composition for the treatment of renal allograft transplant rejection is experimentally shown.

FIELD OF THE INVENTION

The present invention relates to the use of a chemokine receptorantagonist together with a cyclosporin to produce a pharmaceuticalcomposition for treating or preventing rejection of transplanted organs,tissues or cells. It also relates to said pharmaceutical compositionsfor the simultaneous, separate or sequential use of its activeingredients for the above specified therapy.

In particular, it relates to the use of Met-RANTES together withcyclosporin A to produce a pharmaceutical composition for the treatmentof renal allograft transplant rejection.

BACKGROUND OF THE INVENTION

The mechanisms by which a T cell response to a foreign (allogeneic orxenogeneic) protein or cell or organ is mounted are fairly wellunderstood. Antigen presenting cells (APCs) are attracted to areas ofinflammation or damage (that may be induced by surgicaltransplantation). The repertoire of T cells in the periphery isconstantly surveying tissues for evidence of pathogens or the presenceof foreign (allo- or xenogeneic) tissue. Once any of these warningsignals are recognised, the APCs engulf the protein, digest it andpresent it to the host's immune system.

The immune system is well equipped to rapidly identify foreign, diseasedor inflamed tissue and rapidly destroys it. This has always been a majorbarrier to tissue, organ and cell transplantation as well as genetherapy. Major problems are generally associated with chronicinmmunosuppression, encapsulation or immunoisolation. The unwanted sideeffects of chronic immunosuppression include increased susceptibility toopportunistic infection and tumour formation.

In particular, acute renal allograf rejection is mediated by bothalloantigen-dependent and -independent factors and is characterised by amononuclear cell infiltrate consisting mainly of T lymphocytes,monocyte/macrophages and occasional eosinophils (Gröne H. J., 1996,Valente J. F. et al., 1998, Bishop G. A. et al., 1986). The recruitmentof these leukocytes from the peripheral circulation into thetransplanted organ involves a complex interplay between a series ofmolecules expressed on the leukocyte and endothelial surface (Butcher E.C., 1991, Butcher E. C. et al., 1996, Springer T. A., 1994).

The desire for long-term acceptance of grafted tissue in the absence ofcontinuous immunosuppression is a long-standing goal in human medicine.

Chemokines, a large superfamily of structurally related cytokines, havebeen shown to selectively promote the rapid adhesion, chemotaxis andactivation of specific leukocyte effector subpopulations (Springer T.A., 1994, Nelson P. J. et al., 1998, Luster A. D., 1998. Schlöndorff D.et al., 1997).

Chemokines are characterised by a series of shared structural elementsincluding the conserved cysteine residues used to define the C, C—C,C—X—C and C—X₃—C chemokine subgroups (where X represents an interveningamino acid residue between the first two amino terminal proximalcysteines). All of the various biological actions of chemokines appearto be directed through their interaction with a large family ofseven-transmembrane spanning, C-protein coupled receptors (Nelson P. J.et al., 1998, Luster A. D., 1998, Schlöndorff D. et al., 1997). The celltype specific expression of these receptors appears to control asignificant degree, the leukocyte specificity of chemokine action(Nelson P. J. et al, 1998, Luster A. D., 1998, Schlöndorff D. et al.,1997).

The chemokine RANTES (regulated upon activation, normal T-cell expressedand secreted), a member of the C—C chemokine subfamily, is a ligand fora number of chemokine receptors including CCR1, CCR3, CCR5, CCR9 andDARC (Duffy Antigen Receptor for Chemokines) in humans (Nelson P. J. etal., 1998, Luster A. D., 1998, Schlöndorff D. et al., 1997, Nibbs R. J.et al., 1997). RANTES is a potent chemoattractant for T cells,monocytes, natural killer cells, basophils and eosinophils (Nelson P. J.et al., 1998).

Chemokines such as RANTES, are thought to play pivotal roles in thecellular infiltrates that underlie various disease processes. Forexample, RANTES is expressed in vivo in diseases characterised by amononuclear cell infiltrate including, delayed-type hypersensitivity,necrotizing glomerulonephritis, inflammatory lung disease and renalallograft rejection (Schlöndorff D. et al., 1997, Nelson P. J. et al.,1998, Devergne O. et al., 1994, Luckas N. W. et al., 1996, Lloyd C. M.et al., 1997, Pattison J. et al., 1994, Wiedermann C. J. et al., 1993).In studies of human kidneys undergoing acute cellular rejection, RANTESprotein was found localised to mononuclear infiltrating cells, renaltubular epithelial cells and tile endothelium of peritubular capillaries(Pattison J. et al., 1994, Wiedermann C. J. et al., 1993). Since acutecellular rejection is characterised by an intravascular mad interstitialcellular infiltrate consisting of monocyte/macrophages, T lymphocytesand occasional eosinophils, RANTES is potentially a key player in thepathogenesis of acute rejection (Schlöndorff D. et al., 1997, Nelson P.J. et al., 1998, Pattison J. et al., 1994, Wiedermann C. J. et al.,1993).

Based upon these observations a model for the role of RANTES in renalallograft rejection was proposed (Nelson P. J. et al., 1998, Pattison J.et al., 1994, Wiedermann C. J. et al., 1993). Early in rejection, themicrovascular endothelium becomes inflamed, platelets degranulate,releasing RANTES protein that binds to the endothelial surface. Theinflamed renal tubules and endothelial cells produce additionalchemokines including RANTES. The accumulated surface bound chemokinesthen provide directional signals to circulating leukocytes as they rollacross the endothelial surface (Butcher E. C., 1991, Butcher E. C. etal., 1996, Springer T. A., 1994, Nelson P. J. et al., 1998, Pattison J.et al., 1994, Wiedermann C. J. et al., 1993). Leukocytes recognise thesurface bound protein, upregulate integrins, and firmly adhere to theendothelial surface, undergo diapedesis and extravasation. As theleukocytes become activated, they produce additional cytokines andchemokines thus amplifying and propagating the inflammatory response(Nelson P. J. et al., 1998, Pattison J. et al., 1994, Wiedermann C. J.et al., 1993).

Modification of the amino terminus of the RANTES protein candramatically alter its properties (Proudfoof A. E. et al., 1996, Gong J.H. et al., 1996, Simmons G. et al., 1997). The addition of a singlemethionine residue changes the agonist protein into a RANTES receptorantagonist with nanomolar potency (Proudfoof A. E. et al., 1996). Thisantagonist, Met-RANTES, is bioactive in mouse and rat (Proudfootunpublished), and has been shown to suppress inflammation in murinemodels of allergic skin and rheumatoid arthritis and to partiallyinhibit in necrotizing glomerulonephritis (Teixeira M. M et al., 1997,Plater-Zyberk C. et al., 1997, Lloyd C. M et al., 1997).

Cyclosporins represent a group of nonpolar cyclic oligopeptides, havingimnnunosuppressant activity, produced by the fungus Tolypocladiuminflatum Gams and other fungi imperfecti. The major component,cyclosporin A, has been identified along with several other minormetabolites, cyclosporins B through N. A number of synthetic analogueshave also been prepared. Cyclosporin A is a commercially available drug,which has attained widespread clinical application as immunosuppressantin organ transplantation procedures.

The main problem with cyclosporin A has been its nephrotoxicity(Martindale, 1996), characterised by fluid retention, increased serumcreatinine and urea concentrations, a fall in glomerular filtrationrate, and decreased sodium and potassium excretion. In particular, inrenal graft recipients may be difficult to distinguish nephrotoxicityfrom graft rejection.

DISCLOSURE OF THE INVENTION

We have now found that a combined treatment with a chemokine receptorantagonist and a low dose of a cyclosporin results in a reduction of theinflammatory events associated with transplant rejection, as compared totreatment with a cyclosporin alone.

In particular, we have found that Met-RANTES reduced damage to vasculesand tubules and caused a significant reduction of interstitial rejectionin renal allograft transplantation.

Therefore, the main object of the present invention is the use of achemokine receptor antagonist in combination with a cyclosporin toproduce a pharmaceutical composition for treating or preventing therejection of transplanted organs, tissues or cells. The chemokinereceptor antagonist and the cyclosporin can be administeredsimultaneously, separately or sequentially.

Another object of the present invention is, therefore, the method fortreating or preventing the rejection of transplanted organs, tissues orcells by administering simultaneously, separately or sequentially aneffective amount of a chemokine receptor antagonist and an effectiveamount of a cyclosporin, together with a pharmaceutically acceptableexcipient.

An “effective amount” refers to an amount of the active ingredients thatis sufficient to affect the course and the severity of the rejection oftransplanted organs, tissues or cells, leading to the reduction orremission of such pathology. The effective amount will depend on theroute of administration and the condition of the patient.

A further object of the present invention are the pharmaceuticalcompositions containing a chemokine receptor antagonist and acyclosporin, in the presence of one or more pharmaceutically acceptableexcipients, for the simultaneous, separate or sequential administrationof its active ingredients for treating or preventing the rejection oftransplanted organs, tissues or cells.

In case of separate or sequential use of the two active ingredients, thepharmaceutical compositions of the invention will consist of twodifferent formulations, each comprising one of the two activeingredients together with one or more pharmaceutically acceptableexcipients.

“Pharmaceutically acceptable” is meant to encompass any carrier, whichdoes not interfere with the effectiveness of the biological activity ofthe active ingredient and that is not toxic to the host to which isadministered. For example, for parenteral administration, the aboveactive ingredients may be formulated in unit dosage form for injectionin vehicles such as saline, dextrose solution, serum albumin andRinger's solution.

Besides the pharmaceutically acceptable carrier, the compositions of theinvention can also comprise minor amounts of additives, such asstabilisers, excipients, buffers and preservatives.

The administration of such active ingredients may be by intravenous,intramuscular or subcutaneous route. Other routes of administration,which may establish the desired blood levels of the respectiveingredients, are comprised by the present invention.

The combined therapy of the present invention is suitable for treatingor preventing the rejection of any transplanted organ, tissue or cell,but it is particularly advisable in cases of kidney transplantations,due to the nephrotoxicity of cyclosporin A.

The term “chemokine receptor antagonist” means any molecule, which actsas antagonist to the mature full-length naturally-occurring chemokinesand, preferably, does not show significant chemoattractant activity. Forthe measurement of said chemoattractant activity reference is made forexample to (Nelson P. J. et al., 1998).

The chemokine receptor antagonist is preferably selected among truncatedRANTES molecules reported in International patent application WO97/44462, truncated MCP-3, RANTES (missing the first five, six, seven,eight or nine amino acid residues) and MIP-1α described in Internationalpatent application WO 98/06751, truncated RANTES (missing the first twoamino acid residues) and MCP-2 described in European patent applicationNo. 9711663.8 or N-terminally extended RANTES (Met-RANTES, Leu-RANTES,and Gln-RANTES) described in WO 96/17935. Met-RANTES is particularlypreferred. To the above-cited patent applications, reference is madealso for the methods of preparation of the chemokine receptorantagonists mentioned.

The cyclosporin is selected among cyclosporin A, metabolites orsynthetic analogues thereof. Preferably, it is cyclosporin A.

Therefore, a preferred embodiment of the invention consists in thecombined use of Met-RANTES and cyclosporin A for treating or preventingthe rejection of kidney allograft transplantation. In this case, theApplicant has found that it is possible to reduce the effective dose ofcydosporin and this is a great advantage considering the dose-dependenttoxicity to the kidney which is known to be associated with thecyclosporin treatment.

The above effect has been showed with in vivo experiments on rats.

The invention will now be described by means of the following Examples,which should not be construed as in any way limiting the presentinvention. The Examples will refer to the Figures specified here below.

DESCRIPTION OF THE FIGURES

FIG. 1. The ability of RANTES to bind directly to microvascularendothelium before and after 12 hours stimulation with IL-1β (5 ng/ml)was determined. DMVEC was grown on 96 well plates and RANTS measuredusing a modified ELISA procedure.

FIGS. 2 a and b: The effects of Met-RANTES on firm arrest, spreading ortransmigration of MonoMac 6 cells on activated microvascular endotheliumunder physiological flow. DMVEC grown to confluence in Petri dishes werestimulated with IL-1β (5 ng/ml) or left untreated (control) for 12hours, and pre-incubated with or without RANTES (10 ng/ml) for 30minutes. MonoMac 6 cells were pre-treated with or without Met-RANTES (1μg/ml) for 30 minutes, and perfused at a constant flow rate of 1.5dyn/cm². (a) Firm adhesion to DMVEC was determined by counting thenumber of firmly adherent monocytes in multiple fields after a 5 minutesperiod, and expressed as cells/mm². (b) Monocytes undergoing spreadingor transmigration were counted after 5 minutes in multiple high powerfields, and expressed as the percentage of initially firmly adherentcells. Data represent mean±SD of 3 independent experiments. (Note: theresults were reproducible over a range of Met-RANTES from 0.01 to 1μg/ml).

EXAMPLES Materials and Methods

Cells Used

The monocytic tumour cell line MonoMac 6 was cultured in RPMI 1640 with10% FCS supplemented as previously described (Ziegler-Heitbrock H. W .L.et al., 1988). The cells were routinely carried in 24-well plates(Costar) and the media and sera tested for low LPS content. Primaryhuman dermal microvascular endothelial cell (DMVEC) from human neonatalforskin were obtained from Dr. K. Degitz (Dermatology, LMU, Munich,Germany). The cells were carried in MCDB 131 media (Gibco BRL,Eggenstein Germany) supplemented with 10% Fetal calf serum (BoehringerMannheim, Germany), 1 mg/ml Hydrocortisone acetate (Sigma, Deisenhofen,Germany), 5×10⁵ M Dibutyryladenosinemonophosphate (Sigma, Deisenhofen,Germany), 2 mM Glutamine (Seromed, Berlin, Germany), 100 U/mlPenicillin, 100 mg/ml Streptomycin, 25 mg/ml Amphtericin B(antibiotic/Antimycotic Sol. Gibco BRL, Eggenstein, Germany) andincubated at 37° C. and 5% CO2. The cells were grown on T75 flasks, 35mm Petri plates (Costar, Corning, N.Y.) or 96 well flat bottom plates(Nunc, Wiesbaden, Germany) precoated with 0.5% gelatine (Sigma,Deisenhofen, Germany). Medium was changed every 2-3 days. Cells werecharacterised, and purity of cultures was ensured through morphologicappearance and immunofluorescence flow cytometry for CD31 surfaceexpression.

Materials

Materials for histologic studies were obtained from Merck (Darmstadt,Germany). Materials for chemical and immunologic measurements weresupplied by Sigma (Munich, Germany). IL-1β and TNFα were purchased fromSigma (Munich, Germany). Generation of recombinant RANTES and the RANTESspecific monoclonal antibody VL1 were described previously (VonLuettichau 1., 1996). Met-RANTES was produced and endotoxin removed forin vivo studies as described previously (Proudfoof A. E. et al., 1996,Teixeira M. M et al., 1997, Plater-Zyberk C. et al., 1997, Lloyd C. M etal., 1997).

Animals and Renal Transplantation

Inbred male rats were used in all experiments. Lewis (LEW, RT1¹) ratsserved as recipients of Fisher 344 (F344 RT1^(1v1)) or Brown Norway (BNRT1*) kidneys. The animals were purchased from Charles River GmbH,Sulzfeld, Germany. The rats weighed 190 to 250 gm (Lew and F344) and 140to 170 gm (BN) to adjust for ureter diameter. Transplantation wasperformed using a modification of the technique originally described byFisher and Lee (Fisher B. et al., 1965). Briefly, the animals wereanaesthetised by ether-drop anaesthesia, the donor kidney was flushedwith 5 ml of cold 0.91% NaCl (4° C.) with or without 100 μg Met-RANTES.The kidney and ureter were removed en bloc including the renal arterywith a 5-mm aortic cuff and the renal vein with a 3-mm vena cava patch.The kidneys were stored in 0.91% NaCl 4° C.

The donor kidney was transplanted to the abdominal aorta and inferiorvena cava of the recipient animal, below the left renal artery, byend-to-side anastomoses with 8-0 nonabsorbable monofilament nylonsuture. Ureter anastomosis was performed end-to-end with 11-0nonabsorbabie monofilament nylon suture. Total ischemic time of thedonor kidney varied between 30 and 40 min. Hyronephrosis was evaluatedboth macroscopically at time of death and by light microscopy. Allanimals with hyronephrosis were excluded from the experimental groups.The left kidney of the recipient was always removed at the time oftransplantation. In the Fisher to Lewis transplantation the right kidneywas left in plate to have an internal control for the effects ofMet-RANTES. In Brown Norway to Lewis transplantations a bilateralnephrectomy was performed at the time of transplantation.

Experimental Groups:

Experimental groups were as follows:

-   -   Group 1: Fisher 344 kidney into Lewis rat with one endogenous        kidney.    -   Group 1a: with Met-RANTES 200 μg/day for 7 days (n=9)    -   Group 1b: without Met-RANTES for 7 days (n=9)    -   Group 2: Brown Norway kidney into bilaterally nephrectomised        Lewis rat with CyA 2.5 mg/kg BW administrated per day.    -   Group 2a: with Met-RANTES, 50 μg/day for 12 days (n=4)    -   Group 2b: without Met-RANTES for 12 days (n=4)

Cyclosporin A (CyA) (kindly provided by Sandoz, Basel, Switzerland) wasdissolved in olive oil and administered subcutaneously in aconcentration of 2.5 mg/kg BW per day for 12 days, starting 4 hpost-transplantation. Met-RANTES was dissolved in water and adjusted to0.9% sodium chloride and injected once daily intravenously at a dose of200 μg per day in Fisher to Lewis and at a dose of 50 μg per day inBrown Norway to Lewis transplantation experiments.

Serum Analysis

Blood taken from the aorta at the time of sacrifice was analysed forcreatinine, urea, glucose, and bilirubin using an automated serumanalyser. This did not provide information on renal function for theFisher to Lewis model as the transplanted animals had one endogenouskidney, but these measurements were relevant in the Brown Norway toLewis transplant model.

Histology

Organs (lung, liver, kidney, and spleen) were removed under deepanaesthesia. The organs were quickly blotted free of blood, weighed, andthen processed as needed for histology, immnunohistochemistry, or insitu hybridisation. The organs were cut into 1-mm slices and eitherimmersion-fixed in 4% formaldehyde in phosphate buffered saline (PBS) pH7.35, (PBS: 99 mM NaH₂PO₄×H₂O, 108 mM NaH₂PO₄×2H₂O and 248 mM NaCl) for24 h or fixed in methacam for 8 h and embedded in paraffin, or frozen inliquid nitrogen and consequently stored at −80° C. until used forimmunohistochemistry. Light microscopy was performed on 3 μm sectionsstained by periodic acid-Schiff or Goldner-Elastica.

Immunohistochemistry

The monoclonal antibody ED1 (Serotech/Camon, Wiesbaden, Germany) wasused on methacam fixed paraffin embedded tissue (3 μm) to demonstratemonocytes/macrophages. For detection for CD8 antigen expressed oncytotoxic T lymphocytes, monoclonal mouse antibodies were applied tofrozen sections after ice cold acetone-fixation for 5 min(Serotech/Camon, Wiesbaden, Germany). An alkaline phosphataseanti-alkaline phosphatase detection system was applied (Dako, Hamburg,Germany). Controls, omitting the first or second antibody for eachsection tested, were negative.

Morphometry

Vascular injury score: Preglomerular vessels with endothelial damage,thrombus and endothelialitis were assessed as showing no injury (0), amild (1), moderate (2) and severe (3) degree of injury and evaluated inwhole kidney section including cortex, outer and inner medulla. A degreespecific vascular injury index was defined as the percentage of vesselswith the respective degree of injury encountered in a whole kidneysection. Total vascular injury score was calculated as the sum of allvessels, with all degrees of vascular injury, whereby the number ofvessels with degree one, was multiplied by one, that of degree two, by afactor of two, and that of degree three, by a factor of three(Stojanovic T. et al., 1996). Tubular inflammation score: Tubular damagewas evaluated as non-existent (0), mild (1), moderate (2) and severe (3)as judged in 20 High power Fields (HPF) of cortex and cuter stripe ofouter medulla. The total tubular damage score was calculated asdescribed for the total vascular injury score, Interstitial inflammationscore: The extent of interstitial infiltration by mononuclear cells wasjudged as non-existent (0), mild (1), moderate (2) and severe (3) andthe total score calculated as described for the total vascular injuryscore. The number of monocycles/macrophages and T cells within capillaryconvolutes of glomeruli was calculated as the mean of the respectivenumbers in all glomeruli in one kidney section

In Situ Hybridisation

Single-stranded RNA probes were generated by in vitro transcription of acDNA clone of rat RANTES (Dr. H Sprenger, Marburg, Germany). In vitrotranscription was carried out using a Trans-Probe-T kit (Pharmacia,Freiburg, Germany) and digoxigenin-labeled uridine triphosphate(Boehringer, Mannheim, Germany). The vector (pBluescript KS (+)Stratagene, Heidelberg, Germany) was cut with BamHI and transcribed withT3-RNA polymerase to yield antisense probe, to yield sense probe, theplasmid was cut with EcoRI followed by transcription with T7 RNApolymerase. After deparaffinization, kidney sections were digested with20 μg/ml proteinase K (Boehringer) m PBS for 16 min. Sections werepostfixed for 5 min in 4% formaldehyde and acetylated (0.25% aceticanhydride in 0.1 M triethanolamine, 10 min). For in situ hybridisationwith digoxigenin labelled mRNA, the following hybridization buffer wasused: 5×standard saline citrate (SSC), 50% formanide, 50 μg/ml tRNA, 50μg/ml heparin, and 0.1% sodium dodecylsulfate.

After hybridisation at 56° C. for 16 h, slides were washed once in 4×SSCand 2×SSC for 10 min at 37° C., followed by a washing step in 0.55×SSCfor 30 min and 0.1 SSC at 22° C. for 15 min Antidigoxigenin antibodyincubation and alkaline phosphatase reaction was carried out accordingto guidelines by the manufacturer (Boehringer, Mannheim, Germany),taking nitro-blue tetrazolium and 5-bromo-4-chloro-3-indolylphosphate ascolour reagents (Stojanovic T. et al., 1996, Simon M. et al., 1995).

RNase Protection Assay

Total RNA was isolated from whole rat kidney as previously described(Simon M. et al., 1995). RNase protection experiments were performedusing a commercial RPA kit (PharMingen, San Diego, Calif., probe rCK-1).This kit allowed the simultaneous measurement of mRNA species for rat:IL-1α, IL-1β, TNF-α, TNF-β, IL-2, IL-3, IL4, IL-5, IL-6, IL-10 and IFN-γand the housekeeping genes, GAPDH and L32. 20 μg of total RNA was usedfor each determination. The protected samples were run out on a precastgel (Quickpoint™ Rapid Nucleic Acid Separation System used according tothe manufactures recommendations, Novex, San Diego, Calif.). Theintensity of the specific bands were quantitated using a MolecularDynamics Storm 840 Phosphorimager, normalized to L32 gene expression,and averaged over the three animals analysed.

In Vitro Binding Assay

The DMVEC were grown to confluency on coated 96 well flat bottom plates,The resultant endothelial monolayer was either left untreated or treatedwith various concentrations of IL-L1β (0.1 to 5 ng/ml) for 12 h. TheRANTES binding assay was a modification of a previously describedprocedure (Pattison J. et al., 1994, Wiedermann C. J. et al., 1993).Horseradish, peroxidase (HRP) conjugated anti-human-RANTES monoclonalantibody VL1 (0.1 μg) was pre-incubated at 25° C. for 30 min with anexcess of recombinant human RANTES (20 μg/ml) in DMVEC growth media(without supplements). The chemokine-antibody complex was added thenused to assay the relative chemokine binding capacity of themicrovascular endothelium. The endothelial monolayer was gently washed1× with unsupplemented growth media (25° C.) and the chemokine-antibodycomplex was added and incubated at 25° C. for 30 min. The wells werethen washed four times with media without sera at 25° C. The HRPreaction was developed for 5 min or less. The optical density at 406 nmof the plate was determined using an ELISA plate reader. The resultsdemonstrate changes in the binding capacity of the inflamedmicrovascular endothelium for RANTES protein following activation of theendothelial cells. All experiments were performed in quadruplicate andthe results displayed are representative of three separate experiments.

Florescence Activated Cell Sorting (FACS) Analysis

Flow cytometry analysis of dermal microvascular cells (DMVEC) wasperformed essentially as described (Weber C. et al., 1995). Briefly,confluent DMVEC stimulated with IL-Iβ (5 ng/ml), or left untreated for12 h, were trypsinized, reacted with IL-saturating concentrations ofICAM-1 mAb RR1/1 (kindly provided by Dr. R. Rothlein), E-selectin mAb,VCAM-1 mAb (both Serotec), or isotype control for 30 min on ice, stainedwith fluorescein isothiocyanate (FITC)-conjuzated goat anti-mouse IgG(Boehringer Mannheim), and analysed in a FACScan (Becton Dickinson).After correction for unspecific binding, data were expressed as specificmean log fluorescence intensity (sMFI) in channels.

In Vitro Model System of Monocyle Recruitment on MicrovascularEndothelium Under Physiological Flow Conditions.

The interaction of monocytes with DMVEC was studied in laminar flowassays performed essentially as descried (Weber C. et al., 1997, KukertiS. et al., 1997, Piali L. et al., 1998). Briefly, DMVEC were grown toconfluence in 35 mm Petri dishes, and stimulated with IL-1β (5 ng/ml) orleft untreated for 12 h. The plates were assembled as the lower wall m aparallel wall flow chamber and mounted on the stage of an Olympus IMT-2inverted microscope with 20× and 40× phase contrast objectives.Monotypes (MonoMac 6 cells) were cultured as reported (Ziegler-HeitbrockH. W. L. et al., 1988, Weber C. et al., 1993) and resuspended at 10⁶/mlin assay buffer (HBSS) containing 10 mM Hepes/pH 7.4 and 0.5% HAS.Shortly before assay, 1 mM Mg²⁺and 1 mM Ca²⁺ was added. The cellsuspensions were kept in a heating block at 37° C. during the assay andwere perfused into the flow chamber at a rate of 1.5 dyn/cm for 5 min.For inhibition experiments, monocytes were preincubated with Met-RANTESat different concentrations (0.01-1 μg/ml) for 30 min on ice. The numberof firmly adherent cells after 5 rain was quantified in multiple fields(at least 5 per experiment) by analysis of images recorded with a longintegration JVC 3CCD video camera and a JVC SR L 900 E video recorder,and were expressed as cells/mm². The type of adhesion analysed wasrestricted to primary, i.e. direct interactions of monocytes withendotbelium. As an inverse measure of firm arrest, the number of cellsrolling at reduced velocity on endothelium was determined within thelast 30 sec. of the 5 min intervals, and were assessed as the percentageof all interactions in the field. The number of cells spreading ortransmigrating after 5 min intervals was determined in high power fieldsas described (Luscinskas F. W. et al., 1994), and expressed aspercentage of cells firmly attached.

Statistical Analysis

Values are given ms roman +/− SEM. Statistical analysis was performedusing the Mann-Whithney U-Wilcoxon rank sum test. A p value <0.05 wasconsidered as showing a significant difference between two groups.

Results

Allotransplantation of Fisher 344 (F344 RT1^(1v1)) kidneys into Lewis(LEW. RT1¹)

The transplantation of Fisher (344) rat kidneys into Lewis rats in theabsence of immunosuppression resulted in a characteristic mononuclearcell infiltrate and tissue damage by day 7 following surgery.Histological examination showed local mononuclear cell infiltration ofthe intima of preglomerular arteries, and tubular interstitium. Themajor component of this interstitial mononuclear infiltrate consisted ofmonocyte/macrophage cells. The degree of damage to arteries, arterioles,tubules, and the extent of mononuclear cell infiltration of theinterstitium was graded on a scale from non existent (0), mild (1),moderate (2), to severe (3), using a previously described procedurebased upon semiquantitative morphometry (see Materials and Methods).

The effect of Met-RANTES on this process was examined by treatingtransplanted animals with daily intravenous injections of Met-RANTES at200 μg per animal. The initial injection of Met-RANTES was given within1 hour following formation of the vascular anastomosis duringtransplantation surgery. No additional immune suppressive agent wasgiven during the course of the experiment. Light microscopy andimmunohistology showed no obvious effect of Met-RANTES treatment on theendogenous kidney.

During organ transplant rejection, the transplanted organ generallyincreases in weight due to inflammation. The results summarised in Table1, show that the Met-RANTES treated animals had a statisticallysignificant reduction in transplanted organ weight relative to theuntreated animals. The results also suggested a reduction in T cell andmonocyte infiltration of glomeruli, however, this reduction was notconsidered statistically significant (Mann-Whitney U-Wilcoxon rank sumtest). The most profound effects of Met-RANTES treatment are summarisedin Table 2. The data demonstrate a significant reduction in the vascularinjury and tubular rejection score of the Met-RANTES treated animalsrelative to that seen in the untreated animals. While the general trendregarding interstitial rejection score showed an apparent reduction inthe Met-RANTES treated animals, this could not be consideredstatistically significant (Mann-Whithney U-Wilcoxon rank sum test).

Histological sections and immunohistochemical stains were examined toevaluate the effects of Met-RANTES on the rejection process. The kidneyswere removed seven days following transplantation and prepared asdescribed in Materials and Methods.

Vascular damage with mononuclear cells present within the lumen and thewall of arteries were observed in untreated kidneys. In contrast,Met-RANTES treated animals showed no vascular rejection. Theinterstitial region of untreated animals demonstrated infiltration of alarge number of dark staining mononuclear cells within the interstitiumand tubules. By contrast, Met-RATES treated animals demonstrated reducedmononuclear infiltration, less tubular damage with a well-developed redbrush border of proximal tubules.

Localisation of Rat RANTES mRNA by In Situ Hybridisation

Tissue sections taken from rejecting Fisher rat kidneys were used in insitu hybridisation studies to demonstrate cell specific expression ofRANTES mRNA in the rejecting kidney. The results were similar to thosepreviously described for RANTES expression during rejection of humanrenal allografts (Pattison J. et al., 1994, Wiedermann C. J. et al.,1993, Von Luettichau I., 1996). Strong expression by infiltratingmononuclear cells and renal tubules and limited but identifiableexpression by some endothelial cells was seen.

Met-RANTES Treated Animal Show a Reduction in the Expression ofProinflammatory Cytokine mRNA as Determined by RNase Protection Assays

The increased expression of Proinflammatory cytokines such as IL-1α,IL-1β, IL-2-, IL-3, IL-6, TNFβ, TNFα and IFNγ is characteristic of renaltransplant rejection (Nickerson P. et al., 1997. Schmouder R. L. et al.,1995, Strom T. B. et al., 1996, Castro M. D. et al., 1998). Theexpression of these cytokines is an indication of an ongoinginflammatory process. We examined the effect of Met-RANTES on theexpression of a series of cytokine in transplanted Fisher rat kidneysusing quantitative RNase protection assays. Whole organ RNA samples wereisolated from normal control kidneys, untreated transplanted kidneys andMet-RANTES treated transplanted kidneys. The mRNA levels representingthe cytokines: IL-1α, IL-1β, TNFβ, TNFα, IL-2, IL-3, IL-4, IL-5, IL-6,IL-10 and IFNγ, were determined relative to the internal standards L32and GAPDH. The results show that seven days following transplantation,the untreated kidneys upregulated mRNAs coding for IL-1α (24 fold), TNFβ(3.2 fold) and IFNγ (1.7) with the most pronounced increase seen inIL-1β (8.4 fold) and TNFα (4.6 fold). No mRNA expression of IL-2, IL-4,or IL-5 was detected in these kidneys at this time point (7 days posttransplantation). The corresponding Met-RANTES treated animals showed areduced average expression of IL-1α (25%), IL-1β (48%), TNFβ (34%), TNFα(24%) and IFNγ (24%) relative to the untreated animals.

Transplantation of Brown Norway Rat Kidneys into Lewis Rats: Effect ofMet-RANTES in Conjunction with Low Dose Cyclosporin A (CyA).

We then expanded the experiments to determine if Met-RANTES couldcomplement low dose CyA treatment in renal transplant rejection. Forthis procedure, we chose a renal transplant model that would yield amore vigorous rejection episode, namely, the transplantation of BrownNorway kidney into the Lewis rat. A bilateral nephrectomy was performedat the time of transplantation. The level of CyA used, 2.5 mg/kg of bodyweight administered subcutaneously per day, was previously shown not tosignificantly block renal rejection in this model (Gröne unpublishedresults (Stojanovic T. et al., 1996)). Finally, to better detect anysynergistic action, a reduced dose of Met-RANTES, 50 μg per animal perday, was used in these experiments. The results summarised in Table 3show a statistically significant reduction in the vascular and tubulardamage seen in the Met-RANTES/low dose-CyA treated animals as comparedto the animals that were only treated with the low dose-CyA. Inaddition, a significant reduction in mononuclear cell infiltration ofthe interstitial region was seen. These histological observations wereconfirmed by functional measurements where serum creatinine was reducedin the Met-RANTES treated animals relative to the untreated controls(0.98±0.12 vs. 1.42+0.17 mg %, (n=3)).

Direct RANTES Binding and Adhesion Molecule Expression on ActivatedMicrovascular Endothelium

Since a reduction of monocyte infiltration into vascular luminal spacesrepresented a prominent feature of Met-RANTES treatment in bothtransplantation models, we set out to study potential mechanisms forthis effect. In the model of the role of RANTES in renal transplantrejection, it was speculated that RANTES protein, released by activatedplatelets or secreted by locally inflamed tissue, accumulates on thesurface of inflamed endothelium where it may support monocyterecruitment (Nelson P. J. et al., 1998, Pattison J. et al., 1994,Wiedermann C. J. et al., 1993). To study the direct binding of RANTES tomicrovascular endothelium, we examined the capacity of activated dermalmicrovascular endothelium (DMVEC) to sequester RANTES protein using amodification of an assay previously employed to detect endothelialsurface binding of RANTES in tissue sections (Pattison J. et al., 1994,Wiedermann C. J. et al., 1993). An HRP-conjugated mAb specific forRANTES, VL1, was incubated with an excess of RANTES protein, and theresulting complex was added to resting or IL-1β-activated microvascularendothelium grown in 96 well flat bottom culture plates. Using anELISA-like format, the capacity of DMVEC to bind the antigen-mAb complexas opposed to mAb alone was determined. While the microvascularendothelium could bind some RANTES protein without prestimulation, thebinding was greatly increased following prestimulation with theproinflammatory cytokine IL-1α (FIG. 1). The background staining ofuncomplexed mAb to unstimulated or activated endothelium was negligible.

To further characterise the inflammatory activation of microvascularendothelium, the surface expression of molecules involved in monocyteadhesion, i.e. E-selectin and the Ig superfamily members ICAM-1 andVCAM-1, were determined on DMVEC using a previously established flowcytometry procedure (Weber C. et al., 1995). The analysis revealed thatresting DMVEC expressed constitutive surface levels of ICAM-1, howeverlittle VCAM-1 or E-selectin was detected (Table 4). Activation of DMVECwith IL-1 for 12 h resulted in an upregulation of ICAM-1 expression anda marked induction of VCAM-1 and E-selectin surface expression (Table4).

Met-RANTES Blocks the Firm Adhesion of Monocytes to InflamedMicrovascular Endothelium but do not Effect Subsequent Events inDiapedesis.

In an attempt to gain insight into potential mechanisms of action ofMet-RANTES, we studied whether a blockade of RANTES receptors couldinhibit the firm arrest and diapedesis of monocytes on microvascularendothelium. To this end, we used monocytic MonoMac 6 cells that showthe adhesive characteristics and integrin repertoire of maturemonocytes, and express several chemokine receptors including CCR1 (ErlW. et al., 1995). DMVEC were grown to confluence on Petri dishes whichwere either left unstimulated or were activated with IL-1β (5 ng/ml) for12 h. The microvascular endothelium was then tested in a parallel wallflow chamber where the MonoMac 6 cells were perfused through the chamberat a shear rate of 1.5 dyn/cm² for 5 min. Under such physiological flowconditions, MonoMac 6 cells undergo short periods of rolling, and theattachment of a proportion of cells can be readily converted intoshear-resistant arrest. After 5 min of accumulation, the number ofMonoMac 6 cells that had undergone firm adhesion to the endothelium wasdetermined (FIG. 2(a)).

Few monocytic cells firmly adhered to unstimulated microvascularendothelium and the pre-exposure of the endothelial cells to RANTESprotein showed no significant effect. Prestimulation of themicrovascular endothelium with IL-1β resulted in an increase in shearresistant adhesion of monocytes. Inhibition with mAb confirmed previousfindings that this process is mediated by monocyte α4 and β2 integrinsthat interact with ICAM-1 and VCAM-1 expressed on activated endothelium,respectively (Kukerti S. et al., 1997, Luscinskas F. W. et al., 1994).Consistent with the immobilisation of RANTES in direct binding assays,pre-exposure of IL-1β-activated microvascular endothelium to RANTESprotein markedly enhanced the firm arrest and accumulation of monocyteswithin 5 min (FIG. 2 a). Notably, pre-incubation of monocytes withMet-RANTES at various concentrations (0.01-1 μg/ml) completely blockedRANTES-mediated shear resistant adhesion of monocytes on IL-1β activatedDMVEC (FIG. 2 a, and data not shown), in parallel, the fraction ofmonocytes rolling on the activated microvascular endothelium which canbe used as an inverse measure of firm arrest, was reduced afterpreexposure to RANTES, but was restored by Met-RANTES, indicating thatthe number of initial interactions with the activated endothelium wasunaffected. After firm arrest, a fraction of monocytes underwent shapechange or spreading, and some ultimately migrated in-between or underendothelial cells. However, RANTES or Met-RANTES did not alter spreadingor transmigration (FIG. 2 b), inferring the involvement of othersignals. Thus, these results indicate that Met-RANTES may reducemonocyte recruitment during renal transplant rejection by blockingmonocyte arrest to inflamed microvascular endothelium.

TABLES

TABLE 1 Fisher and rat kidney transplanted into Lewis rats. The numberof monocytes/macrophages and T cells within capillary convolutes ofglomeruli was calculated as the mean of the respective numbers in allglomeruli in one kidney section. Control Met-RANTES (n = 9) (n = 9) BodyWeight (g) 211.8 ± 5.15   195 ± 5.98 Transplant-  1.41 ± 0.048  1.15 ±0.08* Kidney Weight Endogenous 0.91 ± 0.04  0.8 ± 0.04 Kidney Weight TCells in 3.98 ± 0.81 2.75 ± 0.45 Glomeruli Macrophages in 9.16 ± 1.695.98 ± 0.87 Glomeruli *Indicates significant (p < 0.05) differencebetween the groups tested.

TABLE 2 Fisher kidney transplanted into Lewis rats. Summary ofhistological and immunohistological analysis of the effects ofMet-RANTES on vascular damage, and interstitial mononuclearinfiltration. Vascular Injury Tubular Damage Interstitial InflammationControl Met-RANTES Control Met-RANTES Control Met-RANTES Grade0 50.67 ±9.02  85.5 ± 2.66 15.56 ± 6.03 46.00 ± 12.15 1.39 ± 1.11 16.00 ± 7.14Grade 1 40.44 ± 5.5  12.8 ± 4.17 32.22 ± 8.25 30.00 ± 6.06  44.17 ±12.39 58.00 ± 8.7  Grade 2 4.44 ± 2.22 0.9 ± 0.6 20.56 ± 5.68 13.00 ±4.1  18.33 ± 5.95   9.50 ± 4.62 Grade 3 5.55 ± 5.55   0 ± 0.0  31.67 ±14.19 8.50 ± 7.46 36.11 ± 14.88 10.50 ± 9.44 SCORE 62.67 ± 18.64 16.1 ±5.2* 33.00 ± 6.44 15.70 ± 5.22  37.78 ± 5.58  24.45 ± 4.62 *Indicatessignificant (p < 0.05) difference between the groups tested.

TABLE 3 Brown-Norway rat kidneys transplanted into Lewis rats. Summaryof histological analysis of the effects of Met-RANTES on vascular andtubular damage, and interstitial mononuclear infiltration in thepresence of low dose CyA. Cyclosporin 2.5 mg/kg b.w./d + CyclosporinMet-RANTES SCORE 2.5 mg/kg b.w./d 50 μg/d VASCULAR INJURY 60.7 ± 1.813.7 ± 7.5*  Tubular Damage 124.3 ± 28.7 28.3 ± 14.8* Interstitial 157.3± 21.3  71 ± 6.1* Inflammation *Indicates significant (p < 0.05)difference between the groups tested.

TABLE 4 Effect of IL-1β on the surface expression of adhesion moleculesin human microvascular endothelial cells. DMVEC were activated withIL-1β (5 ng/ml) or left untreated (control) for 12 hr., and were reactedwith ICAM-1, VCAM-1, E-selectin or isotype control mAbs. The surfaceprotein expression was analysed by FACS in 3 independent experiments andgiven as specific mean fluorescence intensity (sMFI) after correctionfor unspecific binding in channels. SMFI (channels) Control IL-1β ICAM-1Exp. 1 339 502 Exp. 2 386 592 Exp. 3 327 432 VCAM-1 Exp. 1 10 76 Exp. 272 236 Exp. 3 25 129 E-selectin Exp. 1 1 120 Exp. 2 44 265 Exp. 3 28 177

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1. A method of treating or preventing the rejection of transplanted organs, tissues or cells which comprises administering to a subject in need thereof (1) a chemokine receptor antagonist for the RANTES receptors said antagonist selected from the group consisting of Met-RANTES, Leu-RANTES, Gln-RANTES, amino terminally truncated RANTES missing the first two amino acid residues, amino terminally truncated RANTES missing the first five amino acid residues, amino terminally truncated RANTES missing the first six amino acid residues, amino terminally truncated RANTES missing the first seven amino acid residues, amino terminally truncated RANTES missing the first eight amino acid residues, and amino terminally truncated RANTES missing the first nine amino acid residues and (2) a cyclosporin to treat or prevent the rejection of transplanted organs, tissues or cells.
 2. The method according to claim 1, wherein the rejection treated or prevented is renal allograft transplantation rejection.
 3. The method according to claim 1, wherein the chemokine receptor antagonist and cyclosporin are administered simultaneously.
 4. The method according to claim 1, wherein the chemokine receptor antagonist and cyclosporin are administered sequentially or separately.
 5. The method according to claim 1, wherein the chemokine receptor antagonist is Gln-RANTES.
 6. The method according to claim 1, wherein the chemokine receptor antagonist is Leu-RANTES.
 7. The method according to claim 1, wherein the chemokine receptor antagonist is Met-RANTES.
 8. The method according to claim 1, wherein the cyclosporin is cyclosporin A.
 9. The method according to claim 8, wherein the chemokine receptor antagonist is Met-RANTES.
 10. The method according to claim 9, wherein the rejection treated or prevented is renal allograft transplantation rejection.
 11. A pharmaceutical composition comprising at least one pharmaceutically acceptable excipient and the combination of (1) a chemokine receptor antagonist for the RANTES receptors said antagonist selected from the group consisting of Met-RANTES, Leu-RANTES, Gln-RANTES, amino terminally truncated RANTES missing the first two amino acid residues, amino terminally truncated RANTES missing the first five amino acid residues, amino terminally truncated RANTES missing the first six amino acid residues, amino terminally truncated RANTES missing the first seven amino acid residues, amino terminally truncated RANTES missing the first eight amino acid residues, and amino acid residues terminally truncated RANTES missing the first nine and (2) a cyclosporin.
 12. The pharmaceutical composition according to claim 11, wherein the chemokine receptor antagonist is Gln-RANTES.
 13. The pharmaceutical composition according to claim 11, wherein the chemokine receptor antagonist is Leu-RANTES.
 14. The pharmaceutical according to claim 11, wherein the chemokine receptor antagonist is Met-RANTES.
 15. The pharmaceutical composition according to claim 11, wherein the cyclosporin is cyclosporin A.
 16. The pharmaceutical composition according to claim 15, wherein the chemokine receptor antagonist is Met-RANTES. 